Construction of luciferase-expressing pancreatic cancer cell lines and evaluation of their application in detecting cytotoxicity efficacy of immune cells / 中华微生物学和免疫学杂志

Objective:To construct pancreatic cancer cell lines expressing luciferase and mesothelin (MSLN), and evaluate the feasibility of using them as target cells in analyzing the cytotoxicity activity of immune cells.Methods:Lentiviral vectors expressing luciferase and MSLN genes were constructed, and pancreatic cancer cell lines were infected after lentivirus packaging. Single-cell clones were obtained by limited dilution following antibiotic screening, and the stable expression of the target genes were verified. These cells were used as target cells to detect the cytotoxicity of immune cells by real-time cell analysis (RTCA) and luciferase activity. Besides, these luciferase-expressing cells were transplanted into B-NDG mice to establish the animal models of pancreatic cancer, and in vivo optical imaging technology was used to detect the expression of luciferase and monitor the tumor growth in mice. The cytotoxicity of chimeric antigen receptor T (CAR-T) cells was verified in these animal models. Results:Three pancreatic cancer cell lines, panc-1-luc, panc-1-luc-MSLN and capan-2-luc, that could stably express luciferase and MSLN genes were successfully constructed. The expression of the reporter gene in these cells were high, and positively correlated with the number of cells. There were 95.6% of panc-1-luc-MSLN cells expressing MSLN. MSLN-CAR-T cells had specific killing effect on MSLN-positive panc-1-luc-MSLN cells and capan-2-luc cells, with the minimum killing rates of (70.00±18.19)% and (57.00±5.29)%, respectively. But they had no cytotoxicity to MSLN-negative panc-1-luc cells. RTCA results showed that MSLN-CAR-T cells were able to lyse all three pancreatic cancer cell lines, and the minimum killing rates were (56.33±7.64)%, (93.00±2.65)% and (26.33±28.15)%, respectively. The killing of target cells by NK-92MI cells was not depended on MSLN expression. The cytotoxicity in the mice models of pancreatic cancer was consistent with the results in vitro. The in vivo and in vitro test results suggested that the expression of luciferase by target cells could reflect the cytotoxicity of immune cells. Conclusions:This study establishes three pancreatic cancer cell lines stably expressing luciferase, which can be used to evaluate the cytotoxicity of immunotherapy products targeting tumor cells in vitro and in vivo.

β-arrestin1 overexpression suppresses progression of human T-cell acute lymphatic leukemia Molt-4 cell xenograft in mice / 南方医科大学学报

OBJECTIVE@#To investigate the effect of β-arrestin1 overexpression on tumor progression in a NCG mouse model bearing T-cell acute lymphocytic leukemia (T-ALL) Molt-4 cell xenograft.@*METHODS@#Molt-4 cells were tagged with firefly-luciferase (F-Luc) by lentiviral infection, and fluorescence intensity of the cells was detected using a luminescence detector. Molt-4 cell lines with β-arrestin1 overexpression or knockdown were constructed by lentivirus infection and injected the tail vein in sub-lethal irradiated NCG mice. Body weight changes and survival time of the xenografted mice were observed, and the progression of T-ALL in the mice was evaluated using an fluorescence imaging system. Sixteen days after xenografting, the mice were euthanatized and tumor cell infiltration was observed in the slices of the liver and spleen.@*RESULTS@#We successfully tagged Molt-4 cells with F-Luc and overexpressed or knocked down β-arrestin1 in the tagged cells. Bioluminescent imaging showed obvious luminescence catalyzed by F-Luc in Molt-4 cells. After injection of Molt-4-Luc cells into irradiated NCG mice, a gradual enhancement of luminescence in the xenografted mice was observed over time, while the body weight of the mice decreased. Compared with the control mice, the mice xenografted with β-arrestin1-overexpressing Molt-4 cells had significantly prolonged survival time ( < 0.001), while the survival time of the mice xenografted with Molt-4 cells with β- arrestin1 knockdown was significantly shortened ( < 0.001). Histological examination revealed fewer infiltrating tumor cells in the liver and spleen of the mice xenografted with β-arrestin1-overexpressing Molt-4 cells in comparison with the mice bearing parental Molt-4 cell xenografts.@*CONCLUSIONS@#β-arrestin1 overexpression suppresses tumor progression in mice bearing Molt-4 cell xenograft.

Evaluation of Various Anti-oketsu Kampo Formulae—Tokakujokito,Keishibukuryogan and Tokishakuyakusan—for Microcirculation by Live Imaging / 日本東洋医学雑誌

Oketsu is a characteristic pathophysiology in Kampo and traditional East Asian medicine that includes mul­tiple aspects of hemodynamic disorder. Anti­-oketsu drugs or the Kampo formulation used for oketsu show sig­nificant clinical effects on various disorders; however, their underlying mechanisms still remain unclear. We aimed to clarify the characteristics of the pharmacological effects of anti-­oketsu drugs on the microcirculation using a microscopic live imaging technique. Three Kampo formulations, namely tokakujokito, keishibukuryo­gan, and tokishakuyakusan were orally administrated to C57BL/6 mice at a dose of 300 mg/kg diluted in dis­tilled water. Live imaging was performed on the subcutaneous vessels of the mice, including the arteries (di­ameter > 50 μm), arterioles (diameter 10-50 μm) and capillaries (diameter < 10 μm). Tokakujokito widely increased erythrocyte flow velocity and blood flow volume from arteries to capillaries within 60 min of ad­ministration. The effects of keishibukuryogan on the vasodilation of the arterioles were remarkable, and con­tinued up to 120 min after administration. The pharmacological target of tokishakuyakusan was the capillaries, increasing their erythrocyte velocity and blood flow volume;its effect was more slowly expressed than those of the other formulations. Our results clearly demonstrate the sequential and special effects of anti-­oketsu drugs on hemodynamics. These differences may provide pharmacological information on the clinical usage of traditional Kampo formulations.

INTRODUCTION OF DiI DIOLISTIC ASSAY TO LABEL THE NERVOUS CELL AND GLIA / 解剖学报

Objective The diolistic assay has been modified to make it simpler and more efficient in labeling neurons and glia. Methods CNS neurons and glial cells were labeled with DiI diolistic assay in fixed tissue and living brain slices of C57/B6J mice. Results The method allowed the visualization of the fine structure of neurons and glia including synaptic structures such as dendritic spines. Conclusion With the method, the labeling efficacy of cell's fine structure is improved, making it preferable for the analysis of dendritic spine. In addition, the ability to label the living neuron and glia will extend its application vastly.